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Plan A Topics? 1.Making a probiotic strain of E.coli that destroys oxalate to help treat kidney stones in collaboration with Dr. Lucent and Dr. VanWert 2.Making plants/algae that bypass Rubisco to fix CO2 3.Making vectors for Teresa Wasiluk’s project 4.Making vectors for Dr. Harms 5.Cloning & sequencing antisense RNA 6.Studying ncRNA 7.Revisiting blue-green algae that generate electricity 8.Something else? Plan A Assignments? 1.identify a gene and design primers 2.presentation on new sequencing tech 3.designing a protocol to verify your clone 4.presentations on gene regulation 5.presentation on applying mol bio Other work 1.draft of report on cloning & sequencing 2.poster for symposium 3.final gene report 4.draft of formal report 5.formal report Molecular cloning How? 1) create recombinant DNA 2) transform recombinant molecules into suitable host 3) identify hosts which have taken up your recombinant molecules 4) Extract DNA Vectors Solution: insert DNA into a vector General requirements: 1) origin of replication 2) selectable marker 3) cloning site: region where foreign DNA can be inserted Vectors 1) Plasmids 2) Viruses 3) Artificial chromosomes YACs can carry >1,000,000 b.p. contain yeast centromeres to be transmitted at mitosis contain ARS = origins of replication contain telomeres so that don’t lose ends contain a selectable marker (usually a gene for amino acid or nucleoside biosynthesis) Telomere ARS Centromere Selectable marker Foreign DNA Telomere YACs (yeast artificial chromosomes) problems with YACs 1) DNA is unstable • gets deleted • gets rearranged 2) Yeast is hard to work with Telomere ARS Centromere Selectable marker Foreign DNA Telomere Artificial chromosomes 1) YACs (yeast artificial chromosomes) 2) BACs (bacterial artificial chromosomes) • based on the E.coli F’ plasmid • take up to 500 kb • Grow in mutant E.coli that can’t recombine Artificial chromosomes 1) YACs 2) BACs 3) PACs (P1 derived artificial chromosomes) • modified bacteriophage • P1 takes up to 400 kb • much more efficient at infecting hosts Artificial chromosomes YACs,BACs, PACs 4) HACs human artificial chromosomes Molecular cloning Which fragment to clone? Molecular cloning usually no way to pick which fragment to clone solution: clone them all, then identify the clone which contains your sequence • construct a library, then screen it to find your clone Libraries a collection of clones representing the entire complement of sequences of interest 1) entire genome for genomic libraries 2) all mRNA for cDNA Libraries Why? Genomes are too large to deal with: break into manageable “volumes” Libraries How? randomly break DNA into vector-sized pieces & ligate into vector 1) partial digestion with restriction enzymes 2) Mechanical shearing Randomly break DNA Ligate into vector Libraries How? B) make cDNA from mRNA reverse transcriptase makes DNA copies of all mRNA molecules present mRNA can’t be cloned, DNA can Detecting your clone “grow” your library on a suitable host • result • colonies for plasmids or YACs • plaques (clear areas where hosts are dead) for viruses Detecting your clone All the volumes of the library look the same trick is figuring out what's inside usually done by “screening” the library with a suitable probe identifies clones containing the desired sequence Detecting your clone Probes = molecules which specifically bind to your clone • Usually use nucleic acids homologous to your desired clone Detecting your clone Probes = molecules which specifically bind to your clone • Usually use nucleic acids homologous to your desired clone •Sequences cloned from related organisms Detecting your clone Probes = molecules which specifically bind to your clone • Usually use nucleic acids homologous to your desired clone •Sequences cloned from related organisms or made by PCR •http://www.dnalc.org/view/15924 -Making-many-copies-ofDNA.html Detecting your clone Probes = molecules which specifically bind to your clone • Usually use nucleic acids homologous to your desired clone •Sequences cloned from related organisms or made by PCR • Make them radioactive, fluorescent, or “tagged” some other way so they can be detected Detecting your clone by membrane hybridization 1) Denature Detecting your clone by membrane hybridization 1)Denature 2)Transfer to a filter • immobilizes it at fixed location • makes it accessible to probe Detecting your clone by membrane hybridization 1)Denature 2)Transfer to a filter 3) probe with complementary labeled sequences •Will bind your clone Detecting your clone by membrane hybridization 1)Denature 2)Transfer to a filter 3) probe with complementary labeled sequences 4) Detect • radioactivity -> detect by autoradiography • biotin -> detect enzymatically Analyzing your clone FISH (fluorescent in situ hybridization) to metaphase chromosomes to find location of your clone Analyzing your clone 1) FISH 2) “Restriction mapping” a) determine sizes of fragments obtained with different enzymes Analyzing your clone 1) FISH 2) “Restriction mapping” a) determine sizes of fragments obtained with different enzymes b) “map” relative positions by double digestions Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis • used to determine organization & copy # of your sequence Southern analysis 1) digest genomic DNA with restriction enzymes Southern analysis 1) digest genomic DNA with restriction enzymes 2) separate fragments by gel electrophoresis Southern analysis 1) digest genomic DNA with restriction enzymes 2) separate fragments using gel electrophoresis 3) transfer & fix to a membrane Southern analysis 1) digest genomic DNA with restriction enzymes 2) separate fragments using gel electrophoresis 3) transfer & fix to a membrane 4) probe with your clone Northern analysis Similar technique used to analyze RNA Northern analysis 1) Separate by gel electrophoresis Northern analysis 1) Separate by gel electrophoresis 2) transfer & fix to a membrane Northern analysis 1) Separate by gel electrophoresis 2) transfer & fix to a membrane 3) probe with your clone Northern analysis 1) fractionate by size using gel electrophoresis 2) transfer & fix to a membrane 3) probe with your clone 4) determine # & sizes of detected bands Northern analysis determine # & sizes of detected bands • tells size • tells which tissues or conditions it is expressed in Northern analysis determine # & sizes of detected bands • tells size • tells which tissues or conditions it is expressed in • intensity tells how abundant it is